The proximal digestive tract

The proximal digestive tract is the front part of digestive system, which is composed of mouth, salivary gland and esophagus. The mouth is where food goes into the digestive system and where the mechanical digestion begins. After food enters, the teeth are responsible for chewing and grinding food into small pieces, with salivary gland secreting saliva that can soften and moisten food particles. Saliva contains enzymes that can start the digestion of certain ingredients, such as starch.

In the pig transcriptomic analysis, mouth (lip, oral mucosa and tongue), esophagus and salivary gland are included for quantitative RNA measurements, representing the proximal digestive tract. The gene classification strategy highlights genes with an elevated expression in one or a group of tissues compared to all other tissues.

306 genes are classified as mouth elevated out of which 30 genes are highlighted as mouth enriched. Based on the expression in esophagus 108 genes are classified as elevated compared to other tissue types, out of which 3 genes are highlighted as tissue enriched in esophagus. RNA expression levels in salivary gland resulted in 234 genes classified as elevated in salivary gland compared to other tissue types, out of which 14 genes are highlighted as tissue enriched in salivary gland.

The function of the proximal digestive tract tissues is similar between pig and human. Histological image of the pig tissues used in the analysis can be found in the pig tissue dictionary.

Mouth

The mouth is in this study represented by lip tissue, oral mucosa and tongue, and the highest expression value among the three tissues is used as representation of the mouth. The lip tissue was sampled from the bottom lip, the oral mucosa was cut out from the inside lining of the cheek and the tongue was sampled at the body of the tongue, close to the tip where the top layer was pealed of to limit the amount of muscle tissue included in the sample. The tongue is a muscular organ composed of interlacing skeletal muscle, pockets of adipose and connective tissue covered by oral mucosa that includes taste receptors. All mouth tissue types include non-keratinized squamous epithelium.

Gene expression in mouth is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 1 summarizes the number of genes in respective category. In total, 16512 genes are detected above cut of (1NX) in pig mouth. The tissue distribution category highlights 6 genes only detected in mouth while 306 genes are classified as mouth elevated compared to other tissues. Table 1 shows the overlap for the mouth elevated genes and tissue distribution category.

Figure 1, (A) The distribution of all genes across the five categories based on transcript specificity in mouth as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in mouth as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 1, Number of genes in the subdivided categories of elevated expression and tissue distribution in mouth

Mouth elevated expression

The genes with highest tissue specificity score in mouth is the tongue specific keratin 84 (KRT84) as well as carbonic anhydrase 6 precursor (CA6) and uncharacterized protein C6orf15 homolog precursor (C6orf15), both with highest expression in oral mucosa.

Table 2, The 10 genes with highest level of enriched expression in mouth. "mRNA (tissue)" shows the transcript level in mouth as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in mouth and the tissue with second highest expression level.

Esophagus

The pig esophagus is a tube that connects the mouth with the stomach. When food is swallowed, the upper esophageal sphincter will open, allowing food to enter the esophagus, and the epiglottis will fold down to prevent food from going down the larynx and other respiratory organs. Morphologically, the esophagus is similar in human and pig, both of which have non-keratinized epithelium, submucosal glands and similar membrane enzymes. Notably, there are abundant salivary glands in the submucosal layer of pig esophagus, implying a digestive function to some extent, which is different from the human esophagus. Shown in the histological sections in the pig tissue dictionary.

Gene expression in esophagus is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 15498 genes are detected above cut of (1NX) in pig esophagus. The tissue distribution category highlights 1 genes only detected in esophagus while 108 genes are classified as esophagus elevated compared to other tissues. Table 3 shows the overlap for the esophagus elevated genes and tissue distribution category.

Figure 2, (A) The distribution of all genes across the five categories based on transcript specificity in esophagus as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in esophagus as well as in all other tissues. The combination of the two categories is shown in table 3.

Table 3, Number of genes in the subdivided categories of elevated expression and tissue distribution in esophagus

Esophagus elevated expression

The esophagus includes both a thick squamous epithelia, local lymphoid follicles and submucosal secretory glands, similar to salivary gland. The high amount of glands can be seen on the transcriptomic analysis, since esophagus share a high number of group enriched genes with salivary gland and only 3 genes are classified as esophagus enriched, BPIFB2 and BPIFB6 are two of them.

Table 4, The 3 genes classified as enriched in esophagus. "mRNA (tissue)" shows the transcript level in esophagus as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in esophagus and the tissue with second highest expression level.

Salivary gland

The salivary gland is an exocrine gland with the main function to produce saliva. The salivary gland also produces digestive enzymes that break down different nutrients. The main salivary gland is the parotid gland in addition to the sublingual, submandibular gland and numerous smaller salivary glands. In this study the submandibular gland was sampled in all 4 cases. The salivary glands contain both serous and mucous glands as well as ductal cells.

Gene expression in salivary gland is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 3 summarizes the number of genes in respective category. In total, 14937 genes are detected above cut of (1NX) in pig salivary gland. The tissue distribution category highlights 3 genes only detected in salivary gland while 234 genes are classified as salivary gland elevated compared to other tissues. Table 5 shows the overlap for the salivary gland elevated genes and tissue distribution category.

Figure 3, (A) The distribution of all genes across the five categories based on transcript specificity in salivary gland as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in mouth as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 5, Number of genes in the subdivided categories of elevated expression and tissue distribution in salivary gland

Salivary gland elevated expression

In line with the function of the salivary gland, the genes classified as salivary gland enriched are secreted proteins, such as SCGB1D1 and SAL1, as well as STATH coding for the protein that prevents precipitation of calcium phosphate in the saliva.

Table 6, The 10 genes with highest level of enriched expression in salivary gland. "mRNA (tissue)" shows the transcript level in salivary gland as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in salivary gland and the tissue with second highest expression level.

Gene expression in proximal digestive tract compared to other tissues

Proximal digestive tract includes several squamous epithelial tissue types, as well as salivary gland. When comparing the tissue elevated expression in proximal digestive tract with other tissue types the relation to other tissues with squamous epithelium or highly secretory tissues is clear as well as the overlap within the tissues of the proximal digestive tract.

For example esophagus and mouth share the expression of squamous epithelium specific KRT4, while salivary gland and esophagus share several genes classified as group enriched with male glands, such as KCNN4 and CGREF1. CGREF1 is highly expressed in bulbourethral gland. Proximal digestive tract also share the expression of transciption factor PAX9 with male glands and upper respiratory system.

In order to illustrate the relation of proximal digestive tissues to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, compared to all other tissues.

Figure 4. An interactive network plot of the tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows tissues representing the proximal digestive tract. Red nodes represent the number of tissue enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.